Summary This proposal focuses on titin, the largest protein known, in heart function and disease. Titin forms a novel and multifunctional myofilament in the striated-muscle sarcomere with important roles that include regulating the diastolic stiffness of the heart. Recent breakthrough studies revealed that titin is of high clinical importance in both heart failure with preserved ejection fraction (HFpEF), and heart failure with reduced ejection fraction (HFrEF). Although significant progress has been made in understanding the basic biology of titin, major gaps in our understanding still remain, including a mechanistic understanding of how titin causes/contributes to heart disease. An important focus of this proposal will be on titin?s role in diastolic dysfunction, motivated by recent studies on patients with HFpEF that revealed deranged phosphorylation of titin?s molecular spring elements and diastolic stiffening. The full spectrum of posttranslational modifications that occur in HFpEF will be studied and high-resolution time-resolved spectroscopic techniques will focus on uncovering the structural changes in titin?s spring elements triggered by posttranslational modification. Drug screens will focus on identifying compounds that mimic or block these structural changes and functional studies will test whether newly discovered and candidate drugs ameliorate titin-based diastolic stiffening in HFpEF. Post-transcriptional mechanisms will be investigated as well, taking advantage of our recent work that has shown that splicing of titin can be manipulated to upregulate complaint titin isoforms and restore diastolic function. The functional efficacy of identified compounds will be tested on engineered heart tissues as well as on animal models. The second major focus of this proposal will be on titin in HFrEF. Several recent sequencing studies in large groups of patients revealed that mutations in the titin gene (TTN) are causative in ~20% of studied dilated cardiomyopathy (DCM) patients. Many of the mutations are truncation mutations (TTNtv) and they have a preferential location in the A-band segment of titin. The A-band segment is the least well-studied part of titin and an important goal of our research is to critically examine the biology of titin in this region of the sarcomere where disease-causing mutations are prominent. These studies include a focus on the role of titin in interacting with cMyBP-C (cardiac myosin-binding protein C, a clinically important thick filament protein). Animal models will be investigated in which TTNtv have been introduced in different regions of titin?s A-band segment. The effects of the mutations will be studied under baseline conditions, when stressed, and when occurring in combination with mutations in other genes. Importantly, we will also test whether excision of the mutated titin exons ameliorates titin-based DCM. In summary, capitalizing on my >20-year track record of innovative titin research, and utilizing our team of experienced scientists and talented trainees, this proposal sets ambitious goals that are expected to further accelerate understanding of the biology of titin, its role in heart disease and titin?s potential to function as a therapeutic target.